Exploring Aeromonas dhakensis in Aldabra giant tortoises: a debut report and genetic characterization.

Aeromonas dhakensis (A. dhakensis) is becoming an emerging pathogen worldwide, with an increasingly significant role in animals and human health. It is a ubiquitous bacteria found in terrestrial and aquatic milieus. However, there have been few reports of reptile infections. In this study, a bacterial strain isolated from a dead Aldabra giant tortoise was identified as A. dhakensis HN-1 through clinical observation, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS), and gene sequencing analysis. Subsequently, to evaluate its pathogenicity, the detection of virulence genes and mice infection experiments were performed. A. dhakensis HN-1 was found to contain seven virulence genes, including alt, ela, lip, act, aerA, fla, and hlyA. Mice infected with A. dhakensis HN-1 exhibited hemorrhage of varying degrees in multiple organs. The half-maximal lethal dose (LD50) value of A. dhakensis HN-1 for mice was estimated to be 2.05 × 107 colony forming units (CFU)/mL. The antimicrobial susceptibility test revealed that A. dhakensis HN-1 was resistant to amoxicillin, penicillin, ampicillin and erythromycin. This is the first report of A. dhakensis in Aldabra giant tortoises, expanding the currently known host spectrum. Our findings emphasize the need for One Health surveillance and extensive research to reduce the spread of A. dhakensis across the environment, humans, and animals.

Isolation and genomic characterization of one novel goose astrovirus causing acute gosling gout in China.

Novel goose astrovirus (NGAstV) is a member of the genus Avain Avastrovirus (AAstV) and the family Astroviridae. NGAstV-associated gout disease has caused huge economic losses to the goose industry worldwide. Since early 2020, NGAstV infections characterized by articular and visceral gout emerged continuously in China. Herein, we isolated a GAstV strain from goslings with fatal gout disease and sequenced its complete genome nucleotide sequence. Then we conducted systematic genetic diversity and evolutionary analysis. The results demonstrated that two genotypic species of GAstV (GAstV-I and GAstV-II) were circulating in China, and GAstV-II sub-genotype IId had become the dominant one. Multiple alignments of amino acid sequences of GAstV capsid protein revealed that several characteristic mutations (E456D, A464N, and L540Q) in GAstV-II d strains, as well as additional residues in the newly identified isolate which varied over time. These findings enrich the understanding of the genetic diversity and evolution of GAstV and may facilitate the development of effective preventive strategies.

Identification and molecular characterization of novel duck reoviruses in Henan Province, China.

Novel Duck reovirus (NDRV) is an ongoing non-enveloped virus with ten double-stranded RNA genome segments that belong to the genus Orthoreovirus, in the family Reoviridae. NDRV-associated spleen swelling, and necrosis disease have caused considerable economic losses to the waterfowl industry worldwide. Since 2017, a significant number of NDRV outbreaks have emerged in China. Herein, we described two cases of duck spleen necrosis disease among ducklings on duck farms in Henan province, central China. Other potential causative agent, including Muscovy duck reovirus (MDRV), Duck hepatitis A virus type 1 (DHAV-1), Duck hepatitis A virus type 3 (DHAV-3), Newcastle disease virus (NDV), and Duck tembusu virus (DTMUV), were excluded by reverse transcription-polymerase chain reaction (RT-PCR), and two NDRV strains, HeNXX-1/2021 and HNJZ-2/2021, were isolated. Sequencing and phylogenetic analysis of the σC genes revealed that both newly identified NDRV isolates were closely related to DRV/SDHZ17/Shandong/2017. The results further showed that Chinese NDRVs had formed two distinct clades, with late 2017 as the turning point, suggesting that Chinese NDRVs have been evolving in different directions. This study identified and genetic characteristics of two NDRV strains in Henan province, China, indicating NDRVs have evolved in different directions in China. This study provides an insight into the ongoing emerged duck spleen necrosis disease and enriches our understanding of the genetic diversity and evolution of NDRVs.

cPKCγ Inhibits Caspase-9-Initiated Neuronal Apoptosis in an Ischemia Reperfusion Model In Vitro Through p38 MAPK-p90RSK-Bad Pathway.

Strokes are one of the leading causes of death and disability in the world. Previously we have found that conventional protein kinase Cγ (cPKCγ) plays neuroprotective role in ischemic strokes. Further, we found that cPKCγ knockdown increased the level of cleaved (cl)-Caspase-3. However, the precise mechanisms underlying cPKCγ-mediated neuronal death remain unclear. To this end, a model incorporating 1 h oxygen-glucose deprivation/24 h reoxygenation (1 h OGD/24 h R) was established in cortical neurons. We found that cPKCγ knockdown remarkably increased neuronal death after OGD. We also found that cPKCγ knockdown increased the level of cl-Caspase-3 through the upstream initiators Capsases-9 (not Caspase-8/12) in OGD-treated neurons. Overexpression of cPKCγ could decrease neuronal death and cl-Caspase-3 and -9 levels. Moreover, cPKCγ knockdown further reduced the phosphorylation levels of p38 MAPK, p90RSK, and Bad. In addition, the protein levels of Bcl-2 and Bcl-xl were decreased after cPKCγ knockdown, whereas that of Bax was increased. In conclusion, our results suggest that cPKCγ partly alleviates ischemic injury through activating the p38 MAPK-p90RSK-Bad pathway and inhibiting Caspase-9 initiated apoptosis. This may have potential as a therapeutic target for ischemic stroke.

Transcriptome analysis of PK-15 cells expressing CSFV NS4A.

BACKGROUND: Classical swine fever (CSF) is a severe disease of pigs that results in huge economic losses worldwide and is caused by classical swine fever virus (CSFV). CSFV nonstructural protein 4 A (NS4A) plays a crucial role in infectious CSFV particle formation. However, the function of NS4A during CSFV infection is not well understood.  RESULTS: In this study, we used RNA-seq to investigate the functional role of CSFV NS4A in PK-15 cells. A total of 3893 differentially expressed genes (DEGs) were identified in PK-15 cells expressing NS4A compared to cells expressing the empty vector (NC). Twelve DEGs were selected and further verified by RT‒qPCR. GO and KEGG enrichment analyses revealed that these DEGs were associated with multiple biological functions, including cell adhesion, apoptosis, host defence response, the inflammatory response, the immune response, and autophagy. Interestingly, some genes associated with host immune defence and inflammatory response were downregulated, and some genes associated with host apoptosis and autophagy were upregulated. CONCLUSION: CSFV NS4A inhibits the innate immune response, and suppresses the expression of important genes associated with defence response to viruses and inflammatory response, and regulates cell adhesion, apoptosis and autophagy.

Nicotinamide Mononucleotide Adenylyltransferase 1 Regulates Cerebral Ischemia-Induced Blood-Brain Barrier Disruption Through NAD+/SIRT1 Signaling Pathway.

The molecular mechanisms of blood-brain barrier (BBB) disruption in the early stage after ischemic stroke are poorly understood. In the present study, we investigated the potential role of nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1) in ischemia-induced BBB damage using an animal middle cerebral artery occlusion (MCAO) model of ischemic stroke. Recombinant human NMNAT1 (rh-NMNAT1) was administered intranasally and Sirtuin 1 (SIRT1) siRNA was administered by intracerebroventricular injection. Our results indicate that rh-NMNAT1 reduced infarct volume, improved functional outcome, and decreased BBB permeability in mice after ischemic stroke. Furthermore, rh-NMNAT1 prevented the loss of tight junction proteins (occludin and claudin-5) and reduced cell apoptosis in ischemic microvessels. NMNAT1-mediated BBB permeability was correlated with the elevation of nicotinamide adenine dinucleotide (NAD+)/NADH ratio and SIRT1 level in brain microvascular endothelial cells. In addition, rh-NMNAT1 treatment significantly decreased the levels of acetylated nuclear factor-κB, acetylated p53, and matrix metalloproteinase-9 in ischemic microvessels. Moreover, the protective effects of rh-NMNAT1 could be reversed by SIRT1 siRNA. In conclusion, these findings indicate that rh-NMNAT1 protects BBB integrity after cerebral ischemia via the NAD+/SIRT1 signaling pathway in brain microvascular endothelial cells. NMNAT1 may be a novel potential therapeutic target for reducing BBB disruption after ischemic stroke.

cPKCγ ameliorates ischemic injury in cultured neurons exposed to oxygen glucose deprivation/reoxygenation by inhibiting ferroptosis.

Ferroptosis is an iron-dependent pathway of regulated cell death. But the exact mechanism of ferroptosis in ischemic stroke remains unclear. We hypothesize that conventional protein kinase cγ (cPKCγ) can attenuate neuronal death by regulating ferroptosis. In this study, primary cultured cortical neurons were used to establish 1 h oxygen-glucose deprivation (OGD) and reoxygenation (R) 0-12 h (i.e., 1 h OGD/R 0-12 h) as in vitro models of cell ischemia. After 1 h OGD/R 0-12 h, cyclooxygenase 2 (COX2) and acyl-CoA synthetase long-chain family member 4 (ACSL4) levels increased, and glutathione peroxidase 4 (GPx4) levels decreased significantly. Concurrently, GPx4 activity decreases, and iron levels increased. The inhibition of ferroptosis by Liproxstatin-1 ameliorated OGD-induced neuronal injury. Liproxstatin-1 administration prominently induced GPx4 expression and suppressed COX2 expression. Additionally, Liproxstatin-1 administration substantially reduced iron accumulation and rescued GPx4 activity, accompanying by prominent changes in lipid peroxidation indicators. cPKCγ knockdown significantly aggravated neuronal death, and increased GPx4 depletion and COX2 and ACSL4 levels, thus dramatically increasing iron accumulation and GPx4 inactivation. Changes in lipid peroxidation indicators were also significantly increased. Ferroptosis is closely associated with OGD-induced ischemic injury, and cPKCγ can attenuate ischemic injury after OGD via ferroptosis suppression.

Core antigenic advantage domain-based ELISA to detect antibody against novel goose astrovirus in breeding geese.

Goose astrovirus (GAstV), the major causative agent of visceral and joint gout in goslings, is a novel pathogen greatly threatening waterfowl industry. Importantly, the horizontal and vertical transmissibility of GAstV posed a great challenge for disease prevention and control. Given the absence of commercial vaccine, restricting vertical transmission and protecting susceptible goslings must be a priority. Although many detection methods have been established, there is no serological method to detect GAstV-specific antibody, greatly limiting inspection and elimination of infected breeding bird. In this study, the B-cell epitopes of GAstV capsid protein were predicted, and its core antigenic advantage domain (shCAP) was expressed and purified. After authenticating the antigenicity, the recombinant shCAP protein was taken as the coating antigen, and an easily accessible indirect enzyme-linked immunosorbent assay (ELISA) was established to detect GAstV-specific antibody. The working conditions, including antigen concentration, serum dilution and incubation time, blocking buffer concentration, and color developing time, were gradually optimized by checkerboard titration. The cut-off OD450 value of the indirect ELISA for positive sample was 0.379, and the analytical sensitivity was 1:800. There was no cross-reaction with sera against goose parvovirus (GPV), Tembusu virus (TUMV), H5 and H7 subtype avian influenza virus (AIV H5 + H7), and Newcastle disease virus (NDV). The assay was further applied to examine 73 breeding goose serum samples and shared excellent agreement of 93.5% (68/73) with western blot, which also suggested that GAstV is circulating in the goose population in China. In conclusion, the developed indirect ELISA is simple, specific, and sensitive, which will be greatly useful to screen GAstV infection and block vertical transmission. KEY POINTS: • B-cell epitopes of GAstV capsid protein were predicted and expressed as immunogen • A core antigenic advantage domain-based ELISA was established to detect GAstV-specific antibody • The established ELISA will contribute to inspection and elimination of infected breeding geese and provide a useful tool for large scale serological testing of GAstV in geese.

Early Aerobic Exercise Promotes Neurological Function Recovery of Rats after Cerebral Ischemia/Reperfusion by Upregulating the Expression of Heat Shock Protein A5.

OBJECTIVE: The neuroprotective function of heat shock protein A5 (HSPA5) in ischemic stroke has been confirmed. This study aimed to investigate the effects of early aerobic exercise on neurological function recovery from cerebral ischemia/reperfusion and to determine whether these effects are associated with the expression level of HSPA5 in the ischemic penumbra. METHODS: A total of 72 male Sprague-Dawley rats were randomly assigned to the ischemia and exercise group [middle cerebral artery occlusion (MCAO)-Ex, n=18], ischemia and sedentary group (MCAO-St, n=18), sham-surgery and exercise group (Sham-Ex, n=18), or sham-surgery and sedentary group (Sham-St, n=18). The MCAO-Ex and MCAO-St groups were subjected to MCAO for 60 min, whereas the Sham-Ex and Sham-St groups were subjected to an identical operation without MCAO. Rats in the MCAO-Ex and Sham-Ex groups then ran on a treadmill for 30 min once a day for 5 consecutive days. After reperfusion, the motor function of the rats was scored by the Bederson neurological function test, balance beam test, and screen test. Nissl staining was conducted to assess morphological and structural change of nerve cells in the ischemic penumbra. The reverse transcription-quantitative polymerase chain reaction was applied to detect the mRNA expression of HSPA5. Western blot analysis was conducted to determine the protein expression of HSPA5. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining was carried out in the ischemic penumbra after MCAO. RESULTS: Rats receiving early treadmill exercise had lower Bederson neurological function, balance beam, and screen test scores on the 3rd, 7th, and 14th days after MCAO; in addition, more neurons survived in the ischemic penumbra after MCAO, and higher mRNA and protein expression of HSPA5 and fewer TUNEL-positive stained cells were observed. CONCLUSION: Our study demonstrated that early aerobic exercise can improve neurological function recovery after ischemia/reperfusion. Furthermore, the increased level of HSPA5 in the ischemic penumbra might be one of the mechanisms of enhanced neurological function recovery.

Downregulation of miR-338-3p alleviates neuronal ischemic injury by decreasing cPKCγ-Mediated autophagy through the Akt/mTOR pathway.

Ischemic stroke is the leading cause of mortality and disability in aging populations. Dysregulation of microRNA is associated with the pathophysiology of ischemic brain injury. Previously, we found that miR-338-3p was prominently downregulated in OGD-treated neurons, which indicates that miR-338-3P potentially plays an important role in ischemic injury. Furthermore, we performed a bioinformatic analysis and found that conventional protein kinase cγ (cPKCγ), an important autophagy regulator, is a potential target of miR-338-3p, and it is upregulated in neurons after ischemic injury. Therefore, we speculated that miR-338-3P may play a role in neuronal autophagy associated with ischemic brain injury by regulating cPKCγ levels. In the present study, oxygen glucose deprivation was used to test this hypothesis. Our results show that miR-338-3p expression is prominently downregulated after OGD. Additionally, miR-338-3p knockdown attenuated ischemic injury and simultaneously reduced the microtubule-associated protein 1 light chain 3 (LC3)-II/LC3-I ratio, which contributes to neuronal survival after ischemia. Moreover, the cPKCγ protein level increased, and miR-338-3p recognized the 3'-untranslated region of the cPKCγ messenger RNA (mRNA) and negatively regulated the cPKCγ protein level by promoting the degradation of its mRNA. In addition, Lv-cPKCγ blocked the pri-miR-338-3p-induced decrease of the Akt and mammalian target of rapamycin (mTOR) phosphorylation levels, as well as the accompanying increase of the LC3-II/LC3-I ratio, thereby alleviating ischemic injury. This suggests that miR-338-3p downregulation following ischemic injury alleviates neuronal injury by targeting cPKCγ, thereby activating the Akt/mTOR signaling cascade and decreasing downstream autophagy. These results provide a potential therapeutic target for ischemic stroke.

Identification and genomic characterization of emerging goose astrovirus in central China, 2020.

Astroviruses are a non-enveloped virus with large host range breadth. AstV-associated gastroenteritis in human and animal, nephritis in chicken, gout in gosling and hepatitis in duckling pose great threats to public health and poultry industry. Since early 2020, continuous emergence of fatal goose astrovirus (GAstV) infections characterized by articular and visceral gout was reported in China. Here, we described two outbreaks of emerging gout disease in two different goose farms of central China. Two virulent GAstV strains, designated as HNKF-1/China/2020 and HNSQ-6/China/2020, were isolated, and the fifth passage of the isolates could cause urate crystals accumulated in the allantoic fluid and even deposited around great vessels and embryo bodies. Meanwhile, the source of these GAstV outbreaks was tracked to goose hatcheries. The prevalence of GAstV in the goose embryos with hatch failure was confirmed, and embryo-origin HNXX-6/China/2020 was further isolated. The complete genome of these three newly isolates was then sequenced and analysed. The results showed that Chinese GAstVs have formed two distinct groups, and the three GAstV isolates, as well as most of the Chinese GAstVs, belong to the G-I group. There are several amino acid mutations in the three newly identified GAstVs, such as A520T, S535R, V555I and A782T in ORF1a and Q229P in ORF2, suggesting the field stains, HNKF-1/China/2020 and HNSQ-6/China/2020, might derive from the weak goose embryo via vertical transmission. Moreover, the phylogenetic analysis of the complete viral genome and individual viral proteins revealed that Chinese GAstV strains have been constantly evolving towards more complicated and various directions. Our study reported the recently emerging GAstV outbreaks in central China, and further analysed the genetic characteristics of three virulent G-I GAstV isolates from commercial goose farms and goose hatchery, indicating the diverse transmission of the virus and providing a basis for developing effective preventive measures and control strategies.

Isolation and Characterization of a Variant Psedorabies Virus HNXY and Construction of rHNXY-∆TK/∆gE.

The outbreak of pseudorabies in China, caused by more virulent pseudorabies virus (PRV) than the classical strains, has led to considerable economic losses. In this study, PRV strain HNXY was isolated from the Henan province of China in 2015 from the pig farm with severe reproductive failure in sows and a high mortality in piglets. The 50% tissue culture infectious doses (TCID50) of HNXY in Vero cells were examined to be 106.5/mL, and the neutralisation titer against Bartha-K61 was significantly higher than against HNXY when tested with the serum from Bartha-K61 vaccinated pigs. The 50% lethal doses (LD50) of HNXY to six-week-old BALB/c mice and two-month-old PRV-free pigs were both 102.3 TCID50. HNXY was classified as genotype II, and numerous amino acid variations were found in gB, gE, gC, gD, TK, and RR1 proteins, compared with PRV from other countries or those prevalent in China before 2012. The attenuated rHNXY-∆TK/∆gE was further constructed, which presented significantly smaller plaques than HNXY, as well as the similar growth kinetics. rHNXY-∆TK/∆gE was confirmed to be non-pathogenic to six-week-old BALB/c mice and zero-day-old piglets. This study isolated updated PRV promising to develop into a new vaccine candidate.

Development of an improved dual-promoter-based reverse genetics system for emerging Senecavirus A.

Senecavirus A (SVA), a recently emerging picornavirus, poses a great threat to the swine industry because it causes swine idiopathic vesicular disease and epidemic transient neonatal losses. Thus far, the progress in SVA viral pathogenesis studies and vaccine development remains sluggish, and an available and convenient reverse genetics system would undoubtedly promote relevant research. Herein, we established an improved universal dual-promoter reverse genetics system with an SVA-specific hammerhead ribozyme and hepatitis delta virus ribozyme at both terminals of the viral genome; this system could be applied to rescue all SVA strains by both eukaryotic and prokaryotic RNA polymerase systems. The genome of the clone-derived Chinese field strain CH/HeN-2018 was assembled into the universal vector pcDNA-rSVAuni through the Gibson assembly technique. Moreover, two silent mutations, G6848C and C7163 G, were separately engineered into the full-length cDNA clone with one step site-directed mutagenesis to create a KpnI restriction enzyme site, which served as a unique genetic marker. The viruses, designated rCH/HeN-2018-T7, rCH/HeN-2018-CMV, rCH/HeN-2018-6484 m and rCH/HeN-2018-7163 m, were successfully rescued through both CMV- and T7-dependent pathways, and their biological properties were further evaluated. The results showed that all four viruses grew rapidly in PK-15 cells and exhibited viral titers and growth kinetics similar to those of parental wtCH/HeN-2018. The established reverse genetics system is easily operated and can be applied to rescue all SVA strains in a short time, which will be helpful for studying SVA biology, including viral pathogenesis, antiviral therapies and vaccine development.

[Effects of heat shock protein A5 induced autophagy on cerebral ischemia/reperfusion injury in mice].

OBJECTIVE: To determine the role of heat shock protein A5 (HSPA5) induced autophagy on cerebral ischemia/reperfusion in-jury in mice. METHODS: Thirty-six BALB/c mice were randomly divided into sham group, ischcmia/reperfusion (I/R) group, vehicle + I/R group, 3-Methyladenine(3-MA) + I/R group, scramble siRNA group and HSPA5 siRNA + I/R group(n=6). In sham group, the operation was only performed, did not insert line switch. Focal cerebral ischemia was performed using the method of middle cerebral artery occlusion (MCAO) for 60 min and 24 h reperfusion. In vehicle + I/R group and 3-MA + I/R group, 2µl 0.9% NaCl or 3-MA(30 mg/ml) was admin-istered by intracerebroventricular injection 30 min before MCAO; In scramble siRNA + I/R group and HSPA5 siRNA + I/R group, 5µl scram-ble siRNA or HSPA5 siRNA(2µg/µl) was administered by intracerebroventricular injection 24 h before MCAO. Autophagosome in neuron, the expression of microtubule-associated protein light chain 3 (LC3)-Ⅱ/LC3-I in ischemic cortex, the degree of cerebral ischemic injury and neu-rological function score were detected. RESULTS: Initial electron microscopy showed that neuronal morphology appeared to be normal in the sham group. At 24 h after I/R, cell shrinkage, loss of cellular organelles and formation of autophagosomes were observed in the ischemic cerebral cortex of I/R group. In addition, autophagosomes were less frequently observed than that in I/R group. The expressions of LC3-Ⅱ/LC3-I and Beclin-1 protein were increased significantly in I/R group compared with that in sham group(P < 0.05). Compare with I/R group, the LC3-Ⅱ/LC3-I protein levels induced by I/R in 3-MA + I/R group or HSPA5 siRNA + I/R group was decreased effectively (P < 0.05). In addi-tion, the cerebral ischemic injury and neurological symptoms after I/R in 3-MA + I/R group or HSPA5 siRNA + I/R group were exacerbated significantly (P < 0.05). CONCLUSIONS: These results suggest that HSPA5 induced autophagy may play a protective role in focal I/R damage in mice.

Downregulation of miR-181b in mouse brain following ischemic stroke induces neuroprotection against ischemic injury through targeting heat shock protein A5 and ubiquitin carboxyl-terminal hydrolase isozyme L1.

Understanding the molecular mechanism of cerebral hypoxic preconditioning (HPC)-induced endogenous neuroprotection may provide potential therapeutic targets for ischemic stroke. By using bioinformatics analysis, we found that miR-181b, one of 19 differentially expressed miRNAs, may target aconitate hydratase (ACO2), heat shock protein A5 (HSPA5), and ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1) among 26 changed protein kinase C isoform-specific interacting proteins in HPC mouse brain. In this study, the role of miR-181b in oxygen-glucose deprivation (OGD)-induced N2A cell ischemic injury in vitro and mouse middle cerebral artery occlusion (MCAO)-induced cerebral ischemic injury in vivo, and its regulation of ACO2, HSPA5, and UCHL1 were further determined. We found that miR-181b expression levels significantly decreased in mouse brain following MCAO and in OGD-treated N2A cells. Up- and downregulation of miR-181b by transfection of pre- or anti-miR-181b could negatively regulate HSPA5 and UCHL1 (but not ACO2) protein levels as well as N2A cell death and programmed cell death in OGD-treated N2A cells. By using a T7 promoter-driven control dual luciferase assay, we confirmed that miR-181b could bind to the 3'-untranslated rergions of HSPA5 and UCHL1 mRNAs and repress their translations. miR-181b antagomir reduced caspase-3 cleavage and neural cell loss in cerebral ischemic cortex and improved neurological deficit of mice after MCAO. In addition, HSPA5 and UCHL1 short interfering RNAs (siRNAs) blocked anti-miR-181b-mediated neuroprotection against OGD-induced N2A cell injury in vitro. These results suggest that the downregulated miR-181b induces neuroprotection against ischemic injury through negatively regulating HSPA5 and UCHL1 protein levels, providing a potential therapeutic target for ischemic stroke.

Phosphorylated CaMKII levels increase in rat central nervous system after large-dose intravenous remifentanil.

BACKGROUND: Postoperative remifentanil-induced pain sensitization is common, but its molecular mechanism remains unclear. Calcium/calmodulin-dependent protein kinase II (CaMKII) has been shown to have a critical role in morphine-induced hyperalgesia. This study was designed to determine how CaMKII phosphorylation and protein expression levels change in the central nervous system of rats with remifentanil-induced hyperalgesia. MATERIAL AND METHODS: Male Sprague-Dawley® rats were exposed to large-dose (bolus of 6.0 µg/kg and 2.5 µg/kg/min for 2 hours) intravenous remifentanil to induce post-transfusion hyperalgesia. Levels of phosphorylated CaMKII (P-CaMKII) and total protein of CaMKII (T-CaMKII) were determined at different post-transfusion times by Western blot and immunostaining and were compared with controls. RESULTS: P-CaMKII increased significantly (P<0.05) at 0, 0.5, and 2 hours. However, P-CaMKII at 5 to 24 hours and T-CaMKII at 0 to 24 hours post-transfusion did not change significantly in rats' spinal dorsal horn, hippocampus, or primary somatosensory (S1) cortex (n=6 per group). Similarly, immunostaining showed stronger P-CaMKII immunoreactants (P<0.05) and more P-CaMKII- positive cells (P<0.05) in the spinal dorsal horn, CA1 region of the hippocampus, and S1 cortex of rats 0.5 hours post-transfusion compared with the control group treated with 0.9% sodium chloride (n=3 per group). CONCLUSIONS: These results suggest that a temporary rise in the P-CaMKII level in the central nervous system may correlate with remifentanil-induced pain sensitization in the postoperative period.

Increased expression of TNF ligand-related molecule 1A and death receptor 3 in bladder tissues of patients with painful bladder syndrome/interstitial cystitis.

Members of the tumor necrosis factor (TNF) superfamily have been revealed to be associated with painful bladder syndrome/interstitial cystitis (PBS/IC). TNF ligand-related molecule 1A (TL1A) and its receptor, death receptor 3 (DR3), belong to the TNF superfamily and have been implicated in chronic inflammatory diseases. Bladder biopsies from 8 female patients clinically diagnosed with PBS/IC according to the National Institute for Diabetes and Digestive and Kidney Diseases criteria and 8 female bladder carcinoma control patients were investigated to test the protein and mRNA expression levels of TL1A and DR3 using western blotting and real-time RT-PCR. The protein level ratio of TL1A to ß-actin (IC, 0.65±0.03 vs. controls, 0.25±0.02, P<0.001) and of its receptor DR3 to ß-actin (IC, 0.66±0.06 vs. controls, 0.27±0.02, P<0.001) were observed to be significantly higher in the patients with IC. The real-time RT-PCR ΔCts of TL1A minus GAPDH (IC, 7.60±0.52 vs. controls, 10.08±0.32, P<0.001) and the DR3 minus GAPDH (IC, 6.68±0.60 vs. controls, 8.99±0.61, P=0.017) were observed to be significantly lower in the patients with IC, suggesting that the mRNA levels of TL1A and DR3 were higher in the PBS/IC patients. The protein and mRNA expression of TL1A and DR3 are upregulated in the bladder tissues of PBS/IC patients and may be involved in inflammation and apoptosis in PBS/IC.

Identification of differentially expressed microRNAs and their PKC-isoform specific gene network prediction during hypoxic pre-conditioning and focal cerebral ischemia of mice.

We previously reported the involvement of conventional protein kinase C (cPKC) ßII, γ, novel PKC (nPKC) ε and their interacting proteins in hypoxic pre-conditioning (HPC)-induced neuroprotection. In this study, the large-scale miRNA microarrays and bioinformatics analysis were used to determine the differentially expressed miRNAs and their PKC-isoform specific gene network in mouse brain after HPC and 6 h middle cerebral artery occlusion (MCAO). We found 4 up-regulated and 13 down-regulated miRNAs in the cortex of HPC mice, 26 increased and 39 decreased gene expressions of miRNAs in the peri-infarct region of 6 h MCAO mice, and 11 up-regulated and 22 down-regulated miRNAs in the peri-infarct region of HPC and 6 h MCAO mice. Based on Diff Score, 19 differentially expressed miRNAs were identified in HPC and 6 h MCAO mouse brain. Then the miRNA-gene-network of 19 specified miRNAs target genes of cPKCßII, γ and nPKCε-interacting protein was predicted by using bioinformatics analysis of genome databases. Furthermore, the down-regulated miR-615-3p during HPC had a detrimental effect on the oxygen-glucose deprivation (OGD)-induced N2A cell injury. These results suggested that the identified 19 miRNAs, notably miR-615-3p, might target these genes of cPKCßII, γ and nPKCε-interacting proteins involved in HPC-induced neuroprotection.